Verônica Thiemi Tsutae de Sousa
Efeitos do canto de anúncio e do tamanho corporal no
espaçamento entre machos em agregações de
Dendropsophus nanus (Anura, Hylidae)
Dissertação apresentada para obtenção do título de Mestre em Biologia Animal, área de Ecologia e Comportamento Animal, junto ao Programa de Pós-Graduação em Biologia Animal do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de São José do Rio Preto.
Orientador: Prof. Dr. Christopher Gordon Murphy
Co-orientadora: Profª. Drª. Denise de Cerqueira Rossa-Feres
Sousa, Verônica Thiemi Tsutae de.
Efeitos do canto de anúncio e do tamanho corporal no espaçamento entre machos em agregações de Dendropsophus nanus (Anura, Hylidae)/ Verônica Thiemi Tsutae de Sousa. - São José do Rio Preto: [s.n.], 2012.
49 f.: 8 il.; 30 cm.
Orientador: Christopher Gordon Murphy
Co-orientadora: Denise de Cerqueira Rossa Feres
Dissertação (Mestrado) - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas
1. Assunto. 2. Assunto. 3. Assunto. I. Murphy, Christopher Gordon. II. Rossa-Feres, Denise de Cerqueira. III. Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas. IV. Efeitos do canto de anúncio e do tamanho corporalno espaçamento entre machos em agregações de Dendropsophus nanus (Anura, Hylidae).
-Efeitos do canto de anúncio e do tamanho corporal no
espaçamento entre machos em agregações de
Dendropsophus nanus (Anura, Hylidae)
Dissertação apresentada para obtenção do título de Mestre em Biologia Animal, área de Ecologia e Comportamento Animal, junto ao Programa de Pós-Graduação em Biologia Animal do Instituto de Biociências, Letras e Ciências Exatas da Universidade
Estadual Paulista “Júlio de Mesquita Filho”, Campus de São José do Rio
Preto.
BANCA EXAMINADORA
Profª. Drª. Denise de Cerqueira Rossa-Feres
Professor Assistente Doutor UNESP – São José do Rio Preto Co-orientadora
Profª. Drª. Cynthia Peralta de Almeida Prado
Professor Assistente Doutor UNESP – Jaboticabal
Prof. Dr. Itamar Alves Martins Professor Assistente Doutor Universidade de Taubaté
-AO PROF.KIT MURPHY E À PROFA.DENISE PELA OPORTUNIDADE E PELA CONFIANÇA NA
MINHA CAPACIDADE EM DESENVOLVER ESTE PROJETO.
-À FAMÍLIA PELO APOIO AO LONGO DESTA JORNADA.
-AOS AMIGOS DO IBILCE QUE SEMPRE ESTIVERAM DISPOSTOS A AJUDAR.PRINCIPALMENTE
À ESTELA RODRIGUES PINTO, MUITO OBRIGADA!!!!
WOULD SING THEIR SONGS TO THE SILVERY MOON. TENORS SINGERS WERE OUT OF PLACE,
FOR EVERY FROG WAS A DOUBLE BASS. BUT NEVER A HUMAN CHORUS YET
COULD BEAT THE ACCURATE TIME THEY SET. THE SOLO SINGER BEGAN THE JOKE;
HE SANG,“AS LONG AS I’LL LIVE I’LL CROAK, CROAK,I’LL CROAK,”
AND THE CHORUS FOLLOWED HIM:“CROAK, CROAK, CROAK!”
EXERTO DE FROGS IN CHORUS,
RESUMO
Em anuros, a manutenção de espaçamento entre machos constitui uma importante adaptação às agregações reprodutivas, minimizando o mascaramento auditivo e aumentando o sucessoreprodutivo dos machos emissores de vocalização. O canto de anúncio transmite informações com o potencial de influenciar o estabelecimento e a manutenção de espaçamento entre machos, como a habilidade de luta e a localização dos machos. Avaliamos o espaçamento entre machos de Dendropsophus nanus (Anura, Hylidae), que emitem canto de anúncio formado por duas notas: as introdutórias (notas A) e as secundárias (notas B). Testamos três hipóteses: 1) o espaçamento entre machos é influenciado por um ou mais parâmetros do canto de anúncio; 2) o canto de anúncio transmite informações a respeito do tamanho corporal dos machos; 3) o espaçamento entre machos é mediado por notas introdutórias. Machos de D. nanus utilizam ambas as notas para advertir seu tamanho corporal e sua localização no habitat reprodutivo. No entanto, somente as notas A transmitem ambas as informações por meio dos mesmos parâmetros acústicos, relativos à estrutura da nota. As notas B transmitem as informações relativas ao tamanho corporal pela taxa de repetição de notas e de pulsos, enquanto o espaçamento foi influenciado pelo número e duração dos pulsos. Além disso, o espaçamento entre machos foi maior quando mediado por notas A do que por notas B. Assim, as notas A informam a coespecíficos a habilidade competitiva do macho emissor da vocalização, enquanto as notas B advertem a qualidade reprodutiva dos machos às fêmeas.
ABSTRACT
In anurans, the maintenance of spacing between males is an important adaptation to
the chorus, reducing auditory masking and maximizing the transmission of the calls
and males’ reproductive success. Advertisement call conveys information with the
potential to influence the establishment and maintenance of intermale spacing, like
competitive ability and location of males. We evaluate intermale spacing in natural
aggregation of Dendropsophus nanus (Anura, Hylidae), whose advertisement call is
constituted by two notes: the introductory (type A notes) and the secondary (type B
notes). We tested three hypotheses: 1) intermale spacing is influenced by one or more
parameters of the advertisement call; 2) the advertisement call contains information
about male body size; 3) intermale spacing is mediated by introductory notes. Males of
D. nanus use both notes to advertise their body size and their location in the
reproductive habitat to other conspecifics. However, only A notes convey both kinds of
information through the same acoustic parameters, which are related to note structure.
Type B notes advertise body size through note and pulses repetition rate, while spacing
is associated to the number and duration of pulses. Furthermore, intermale spacing
was greater when mediated by A notes than when mediated by B notes. Therefore, A
notes advertise mainly a male’s competitive ability in male-male interactions, while B
notes appear to advertise the males’ reproductive quality to females.
Keywords: Anura, Bioacustics, Intermale spacing, Advertisement call, Acoustic
SUMÁRIO
Introduction ... 10
Material and methods ... 13
Study area ... 13
Study species ... 13
Relationship among advertisement call parameters, body size and male spacing ... 14
General procedures ... 14
Bioacustics analysis ... 15
Relationship between note type and male spacing ... 16
Statistical analysis ... 18
Results ... 20
Discussion ... 23
Tables ... 30
Figures ... 36
INTRODUCTION
Males of many anuran species aggregate in choruses of highly variable density
where they emit advertisement calls to attract females and mediate aggressive
interactions (Wells 1977, Duellman and Trueb 1986, Brenowitz and Rose 1994,
Gerhardt 1994, Haddad 1995). In these aggregations, animals must be able to detect
and recognize conspecific signals, localize signalers, discriminate signal types and
signalers, and extract information from signals and interactions among multiple con-
and heterospecific signalers (Gerhardt 1992, Gerhardt and Bee 2007, Wells and
Schwartz 2007). However, high level of noise, mostly biological (sounds from other
animals), impairs the detection of a signal and the transmission of its information (e.g.
Forrest 1994, Wollerman & Wiley 2002, Bee 2008, Bee and Micheyl 2008, Richardson
and Lengagne 2009).
In anurans, the maintenance of spacing between males may reduce auditory
masking from the sounds of the chorus, therefore maximizing the transmission of the
calls and males’ reproductive success (e.g. Brenowitz et al. 1984, Schwartz 1987,
Ryan 1988, Rose and Brenowitz 1991, Bee 2007, Bee and Micheyl 2008). For
example, Richardson and Lengagne (2009) investigated the effect of spacing on
females’ ability to discriminate among competing calling males within a general high
background noise setting. Through phonotaxis experiments with Hyla arborea, the
authors demonstrated that the discrimination of more attractive male calls by females
was improved with the increase of spatial separation of speakers. Although it is
possible that an optimum pattern of spacing exists, with males occupying calling sites
from where signals emitted by near conspecifics are just barely audible (Brenowitz et
al. 1984, Brenowitz and Rose 1994), which would make competition weaker and
information transmission more effective, such optimal spacing is rarely possible
spacing (Brenowitz et al. 1984, Gerhardt et al. 1989, Dyson & Passmore 1992,
Shepard 2002).
Several studies of male-male interactions demonstrated that males may adjust
their calling behavior in response to the proximity and vocalization of neighboring
conspecifics either by changing the timing of calls or by changing the structure of the
call (rate, duration, or complexity) (e.g. Bosch and Márquez 1996, 2001, Schwartz
2001, Marshall et al. 2003, Owen and Gordon 2005, Tárano and Fuenmayor 2009,
Bates et al. 2010). Yet most of the research on intermale spacing has focused on the
examination of the relationship between spacing and one advertisement call trait: call
amplitude (e.g. Brenowitz et al. 1984, Telford 1985, Gerhardt et al. 1989, Rose and
Brenowitz 1991, Stewart and Bishop 1994, Murphy and Floyd 2005). Since amplitude
attenuates with increasing distance, this trait potentially allows the receiver to assess
the distance of the competing male in the chorus: comparatively, the call of a nearer
male will be perceived by the receiver as having the higher amplitude, whereas the call
of a distant male will be perceived as having a lower call amplitude (e.g. Brenowitz and
Rose 1994, Robertson 1984).
Males are able to mediate spacing through vocalizations. Advertisement calls
traits convey diverse information about the signaler that might influence the spatial
distribution of anuran males, such as signaler’s body size: as the competitive ability
and the outcome of a fight between two individuals are highly dependent on the body
size (Parker 1974, Robertson 1986, Wagner 1992, Bee et al. 1999, Bee and Gerhardt
2001), the assessment of an opponent’s size is an important piece of information for
males to determine from the signals of rivals. Body size may be assessed through
dominant or fundamental frequencies, amplitude, call duration, number of pulses, and
pulse repetition rate (e.g. Robertson 1986, McClelland et. al. 1996, Bee et al. 2000,
Poole and Murphy 2007). Additionally, males of many anuran species may also be able
to mediate spacing through the production of two distinct note types (Littlejohn and
convey separate messages to males and females: the introductory notes often convey
aggressive messages to competing males, while secondary notes function primarily to
attract conspecific females (e.g. Narins and Capranica 1976, 1978, Littlejohn and
Harrison 1985). Advertisement calls consisting of two or more notes are common
among some groups of hylids (Wells 1988) such as Dendropsophus microcephalus
species group. This group includes Dendropsophus nanus, a South American hylid frog
whose advertisement call is formed by two simple pulsed notes: the introductory (type
A notes) and the secondary (type B notes) (Martins & Jim 2003). Martins and Jim
(2003) hypothesized that these notes perform distinct functions: as A notes are emitted
more frequently in the beginning of the chorus activity, when few males are calling, they
should be responsible for establishment and maintenance of intermale spacing, while B
notes would function to attract females as males start to emit this notes after they
establish a calling site.
In this study, we evaluated the intermale spacing between nearest males of D. nanus. Three hypotheses were tested. The first one is that spacing between males of D. nanus in the chorus is influenced by one or more parameters of the advertisement
call. This hypothesis predicts that one or more of these parameters should be
correlated with the distance between calling males and their nearest neighbors. The
second hypothesis is that the advertisement call contains information about male body
size. This hypothesis predicts that one or more parameters of calls should be
correlated with male body size. To test these hypotheses, we conducted a field study in
which we measured parameters of calls and intermale spacing in natural aggregations.
The third hypothesis is that intermale spacing is mediated by introductory notes. We
tested this hypothesis by conducting a playback experiment, in which measured the
nearest-neighbor distance to speakers broadcasting either synthetic A or B notes. It
was expected that the distance between the speaker and the nearest male would be
longer for the speaker broadcasting synthetic A notes than the speaker broadcasting
MATERIAL AND METHODS
Study area
Fieldwork was carried out during the rainy and anuran breeding season, from
January to March 2010 and from December 2010 to March 2011, on farmland located
in Macaubal (20°44’29”S; 49°56’07”W), northwestern São Paulo State, Brazil. The
region’s climate is Aw Köppen-Geiger climate type, which is characterized by hot
and wet summer (October to March) and dry winter (April to September) (CEPAGRI
2010). The annual rainfall varies from 1200 to 1650 mm (Carvalho and Assad 2005),
and the onset of the rainy season varies each year (Rossa-Feres and Jim 2001). The
original vegetation cover of Mesophitic Semideciduous Forest (Atlantic Forest Domain)
with patches of Cerrado (Ab’Saber 2003) was intensively deforested for agricultural
activities, and the remaining fragments of original vegetation are few and small (São
Paulo 2000; Rodrigues et al. 2008).
Study species
Dendropsophus nanus is widely distributed throughout South America, from
Northeastern Brazil southward through central Paraguay, northern Argentina, eastern
Bolivia, to extreme southern Brazil, Uruguay, and La Plata Basin in Argentina (Frost
2011). D. nanus is found only in open areas and is very abundant in the study region,
breeding in both temporary and permanent water bodies throughout the rainy season
(Menin 2002, Vasconcelos and Rossa-Feres 2005). Males call from the ground,
shrubs, grasses, and cattails (Bernarde and Kokubum 1999, Menin 2002, Vasconcelos
and Rossa-Feres 2005, present study). The advertisement call of D. nanus consists in
two notes (type A and type B; Figure 1) emitted in consecutive series. Notes have four
to five spectral peaks (the fundamental and three or four upper harmonics) and the
(fundamental frequency). Harmonics are less distinctive in A notes than in B notes
(Figures 2). Both notes differ significantly (p < 0.05) in their temporal structure (Table
1): type A notes are longer, with lower note repetition rate (notes/second), higher pulse
number, lower pulse duration and higher pulse repetition rate (pulses/second) than type
B notes. Notes also differ (p < 0.05) in sound pressure level (root-mean-square sound
pressure level [RMS SPL], re 20 μPa) and minimum frequency (Table 1), but do not
differ (p > 0.05) in both fundamental and maximum frequencies.
Relationship among advertisement call parameters, body size and male spacing
General procedures
Two permanent (a 9498 m2 lake and a 1074 m2 pond) and one temporary (19
m2) water bodies were visited weekly. Males started to call shortly after sunset, and the
sampling was initiated 30 minutes after the beginning of calling activity, when the
chorus was already stabilized and males were emitting both A and B notes. The
sampling period each night lasted about four hours, and males were selected for
recording at random. Fifty notes of each the calling males (n = 102) were recorded at a
sampling rate of 44100 Hz, 24 bits/sample in mono pattern, and in wave format (.wav)
using a digital recorder Edirol R-09HR coupled to a directional Sennheiser ME66/K3U
microphone positioned at a distance of 1 meter from the male. Air temperature and
humidity were measured with a digital thermohygrometer Minipa MT-240; recordings
were made at a temperature of 23.35 ± 1.14°C (mean ± SD) and relative humidity of
82.77 ± 4.22%. The angle at which the microphone was positioned in relation to the
snout of the calling male was registered; this angle varied from 0 to 280°. To avoid
recording the same male repeatedly within a night, calling sites were marked with a
numbered stake to allow identification of the focal male throughout the sampling period.
After the recording, each male was photographed with an Olympus μ 790 SW
pattern of marks on his back (Figure 3), and we compared these patterns (location,
shape and size) using the software program I³S Manta 2.1 (Hartog & Reijns 2008). In
this software, the user identifies the most distinguishing marks of each image and
stores this pattern in the database. To identify a previously photographed individual, the
pattern of marks of a new image is matched with the patterns of all the known animals
in the database and a ranked list of images is presented. We then compared the new
image with the images ranked by the software to determine if a match existed. If a
match did not exist, the image was entered into the database as a new individual.
Photos and the software program ImageJ 1.42q (Rasband 2009) were used to
obtain snout-vent length (SVL) as a measure of male body size. A ruler was positioned
beside each male when his photo was taken, and the ruler provided a known distance
from which we were able to define a spatial scale in pixel per measurement unit. The
software used the defined scale to calculate the SVL in calibrated units (for example,
centimeters). We adopted this approach, rather than measuring males directly, to avoid
disturbing the males and causing them to retreat from their calling sites.
After recordings, intermale spacing was measured as the distance between the
focal male and his nearest neighbor. Focal male perch height were also determined
because perch height influences sound propagation, with sounds attenuating more
severely when emitted near ground than when emitted from elevated perches (e.g.
Greer & Wells 1980, Wells and Schwartz 1982, Mitchell and Miller 1991); degradation
of the quality of a call may influence the effectiveness of a males’ calls in maintaining
spacing. The total number of males calling at the peak activity of the chorus was
determined each sampled night because average distances between males
necessarily decrease with increasing density of calling males (Brenowitz et al. 1984,
Gerhardt et al. 1989, Dyson & Passmore 1992, Shepard 2002).
Advertisement calls were analyzed using Raven Pro 1.3 (Cornell Bioacoustics
Research Program, 2008). Oscillograms (Figure 4), spectrograms (Figure 5) and power
spectrum views (Figure 6) were generated to measure the following parameters for A
and B notes: note amplitude (sound pressure level, SPL), minimum and maximum
frequency of the note (the frequency at the beginning and at the end of the note,
respectively), fundamental frequency (the lowest harmonic in the frequency spectrum),
dominant frequency (frequency containing the greatest energy), note duration (time
from the beginning to the end of a note), note repetition rate (number of notes per
second), number of pulses per note, pulse duration (time from the beginning to the end
of a pulse) and pulse repetition rate (number of pulses per second). As fundamental
and dominant frequencies coincided, we will refer to these frequencies just as
fundamental frequency from now on. SPL was calculated from RMS Amplitude
measures obtained from Raven of a 500 Hz tone broadcast by a speaker and recorded
for 30 seconds with the digital recorder and the microphone positioned at 1 meter
distance. Recordings were made for SPL values varying from 70 to 100 dB, and
measures were made in intervals of 3 dB SPL with a RadioShack Sound Level Meter
33-2055SPL. A calibration curve was constructed from these measures by fitting an
exponential function (y = 33.698e0.1129x, where x represents SPL values and y
represents RMS amplitude values). With this equation it was possible to back-calculate
SPL values from the RMS amplitudes measured from D. nanus advertisement calls.
Relationship between note type and male spacing
To test the hypothesis that the intermale spacing is mediated by A notes, we
synthesized both A and B notes utilizing the average values of acoustic parameters
obtained from the bioacoustic analysis of D. nanus natural calls recorded during the study’s first rainy season. All values used represent the average for the population for
that parameter. The A note lasted 35 ms and consisted of twelve pulses and four
fourth harmonics attenuated relative to the fundamental by -31.77, -40.86, -45.28 dB.
The B note lasted 19 ms and consisted of six pulses and four harmonics, with a
fundamental frequency of 4.32 kHz and the second through the fourth harmonics
attenuated relative to the fundamental by -33.38, -38.92, -45.30 dB. Synthetic calls
were produced at a sampling rate of 44.10 kHz and depth of 24 bits/sample in mono
pattern with the software Audacity 1.2.6 and were stored as digital files (.wav) on the
digital recorder.
Synthetic calls were played from the recorder through an amplifier and
broadcast from two speakers placed randomly in the study water bodies. We broadcast
synthetic calls at two different alternated sequences, one was constituted by A notes
and the other by B notes, following the natural temporal properties of calling activity.
Because note repetition rate was correlated to the temperature in natural choruses (r =
0.39, p < 0.05), the specific note repetition rate was obtained from the regression of
repetition rate on temperature. Three value of temperature were selected for the
playback generation of repetition rates, representing the central (24.5°C) and end
points (22.5 and 26.5°C) of the range of natural temperatures: 1) for temperature close
to 22.5°C, notes were repeated at a rate of 0.85 notes/s, 2) at 24.5°C, the note
repetition rate was 1 note/s, and 3) at 26.5°C, 1.10 notes/s were emitted from
speakers. When conducting playbacks the note repetition rate selected was the one
whose temperature was the most similar to the temperature at the study site at the
beginning of the playback experiment.
The playback experiment tested the spacing of arriving males relative to
established residents, with speakers simulating resident males. Playback began with
sunset, before males arrived at the study water body each night. The experiment was
conducted on 20 nights (10 nights in each of the two permanent water bodies of water)
and was carried out until the chorus stabilized. Each night the two note types were
randomly assigned to the two speakers, with one speaker broadcasting note type A and
environmental variables (e.g. temperature) that might affect male responses. During
the experiment, the distance between the speaker and the nearest male was
determined with the use of a tape measure (to the nearest 1 mm). We recorded the
positions of calling males relative to the speakers every 15 minutes. Also, we counted
the total number of calling males during the censuses as intermale spacing varies with
chorus density.
Statistical analyses
Because recorded males were identified individually, each male (n = 100) was
treated as an independent subject. For males recorded multiple times, only the first
recording was used. Acoustic data were expressed as the mean calculated from the 30
calls analyzed for each male. Although recordings were made after chorus stabilization,
two of the 102 recorded males only emitted type A notes and were therefore excluded
from analyses. Analysis of variance (ANOVA) was applied to verify whether differences
in number, perch height and distance between calling males existed between
permanents and temporary ponds. Data were normally distributed, and all the statistical
analyses were conducted in R 2.13.2 (R Development Core Team 2010).
To check for significant associations (p < 0.05) between the analyzed acoustic
parameters and microphone angle, air temperature and relative humidity, we calculated
Spearman rank correlation coefficients (r). Humidity was significant associated with
duration (r = 0.24, p = 0.03) and number (r = -0.23, p = 0.03) of pulses of type A notes,
and duration (r = 0.22, p = 0.05), fundamental frequency (r = 0.22, p = 0.04), and
maximum frequency (r = -0.22, p = 0.05) of type B notes. Microphone angle was
significantly associated with fundamental frequency (r = -0.22, p = 0.05) and maximum
(r = -0.27, p = 0.01) frequencies of B notes. Because of these associations, observed
values of acoustic parameters’ were converted to residual values, which were obtained
microphone angle). These residual values were used in subsequent statistical
analyses.
PCA based on correlation matrix (Tables 2 and 3) was applied to reduce the
dimensionality of the data (Budaev 2010, Legendre & Legendre 1998). Through PCA,
highly correlated variables are linearly combined and represented by statistical
variables or Principal Components (PCs). Variables highly correlated (i.e. presenting
high loadings) with the same PC may be considered as having the same cause.
Loadings were considered significant (p < 0.05) when they exceeded ± 0.55 (Hair et al.
2009). Communalities were calculated as the sum of the squared loadings of a variable
in relation to each PC and show how much variance of a variable is explained by the
factorial solution, i.e. the PCs taken together (Hair et al. 2009).
To examine whether the data were appropriate for the application of Principal
Component Analysis (PCA), two tests were applied: 1) the Kaiser-Meyer-Olkin (KMO)
index, which measures the sampling adequacy and should be greater than 0.5 for a
satisfactory factor analysis to proceed (Hair et al. 2009, Budaev 2010), and 2) Bartlett's
sphericity test to verify whether the correlation matrix is an identity matrix, which would
indicate that the factor model is inappropriate (Bartlett, 1937; Hair et al. 2009, Budaev
2010). Results from Bartlett’s sphericity test rejected the hypotheses of null correlation
(type A notes: χ2= 501.75, df = 36, p = < 0.00001; type B notes: χ2= 440.93, df = 36, p =
< 0.00001) and KMO indicated that the sample size is appropriated for PCA (type A
notes = 0.602, type B notes = 0.55).
The number of Principal Components (PCs) to be retained was determined with
Horn’s Parallel Analysis (PA; Horn 1965), which contrasts eigenvalues produced
through a PCA on a number of random data sets of uncorrelated variables with the
same number of variables and observations as the observational dataset to produce
eigenvalues for components that are adjusted for the sample error-induced inflation
according to the Kaiser’s rule must be retained (Horn 1965, Franklin et al. 1995, Dinno
2009). PA was applied using a 95% confidence interval.
We used multiple regression analysis to assess the statistical significance of
each retained component as a predictor of (1) distance among calling males and (2)
male body size. The total number of calling males in each sampled night and the perch
height of recorded calling males were included in each analysis as covariates. From a
global model, i.e. one that incorporates all variables of interest, were generated
submodels by the exclusion of independent variables that were non-significant (p >
0.05), i.e. in each model, variables with the highest value of p were subsequently
excluded until the model was composed only by significant variables. To compare the
submodels and identify the one that best fit the observed data, a second-order Akaike
Information Criteria (AICc) was used; the best fitting submodel is the one with the
smallest value of AICc (Burnham and Anderson 2002).
To evaluate whether A notes function to maintain intermale spacing, we included
the distance between the speaker and nearest calling male as the response variable,
with the note type (A or B) as a within-night factor, the water body in which the playback
experiment was conducted as a between-night factor, and the number of calling males
as a covariate in a repeated measures statistical analysis. We applied Linear Mixed
Effects Model, in which the night was considered a random component, while the note
type, water body, and number of calling males were considered fixed components of
the analysis. As a significantly difference in number of males was detected between the
two bodies of water, we also tested the interaction between the variables. The
assumptions of normality, homocedasticity and sphericity were violated, so we assume
a more conservative significant value (p < 0.01).
RESULTS
Calling males of Dendropsophus nanus had a body size of 21.10 ± 2.20 mm
among water bodies (F2,97 = 8.13, p < 0.001): intermale spacing in the permanent water
bodies was significantly higher than in the temporary pond (post-hoc Tukey test, p <
0.001; Table 4). The number of calling males in each sampled night was highly variable
and different among water bodies (F2,97 = 12.76, p < 0.0001). More males were
recorded at the lake than the permanent pond, and more were recorded at the
permanent pond than at the temporary pond. The lake averaged more calling males
per night than did the permanent pond (post-hoc Tukey test, p < 0.0001; Table 4) but
no difference was detected between the permanent and temporary ponds (post-hoc
Tukey test, p > 0.05). Differences among bodies of water were also detected in perch
height (F2,97 = 11.68, p < 0.0001): calling males adopted lower perch height in
temporary water bodies, where only grasses were available to be used as calling sites,
than in permanent water bodies (post-hoc Tukey test, p < 0.001; Table 4), where
shrubs, grasses and cattails were available for perches.
PCA for the acoustic parameters of type A notes reduced the original nine
variables to four factors that accounted for 75.51% of the observed variation (Table 5).
PC1 represented parameters associated with the structure of the note emitted by the
signaler: duration of notes, number of pulses, and pulse repetition rate loaded
positively, and duration of pulses loaded negatively. Taken together, parameters
grouped by this factor indicated that males produced longer notes by increasing the
number of pulses while simultaneously shorting the duration of each pulse. PC2 was
negatively associated with maximum frequency and positively associated with note
repetition rate (Table 5), indicating that males will call at higher rates when maximum
frequency is lower. PC3 grouped fundamental and minimum frequencies, both of which
were positively associated with this factor (Table 5). PC4 was associated positively with
SPL (Table 5). These four factors were then renamed accordingly to the parameters
that loaded significantly onto these factors: Note Structure (= PC1), Note Rate (= PC2),
Frequency (= PC3) and Amplitude (= PC4). We used these factors as the predictor
For B notes, five PCs were retained, and they explained 85.73% of the variance
(Table 6). PC1 grouped two acoustic parameters with significant loadings: duration and
number of pulses. The first is negatively, and the second is positively associated with
PC1 (Table 6); i.e. the B note consists of a high number of short pulses. Balancing the
number and duration of pulses, males determine the structure of the note emitted. PC2
was positively associated with repetition rate of notes and pulses (Table 6). PC3 was
positively associated with the minimum frequency. PC4 was significantly associated
with SPL and maximum frequency. Duration of notes was positively associated with
PC5. These factors were renamed as: Note Structure, Repetition Rate, Minimum
Frequency, Amplitude, and Note Duration, respectively.
Regression models to assess the statistical significance of each PC for note
type A as a predictor of distance between calling males showed that, although both
PC1 (Note Structure) and PC2 (Note Rate) components were incorporated to the best
fit model (R = 0.23; Adjusted R² = 0.20; F4,95 = 7.31; p < 0.0001; AICc: 91.45; AICc
weight: 0.344), only the first PC was statistically significant (Table 7). This result
indicates that males who produced type A notes with fewer, longer pulses within shorter
notes were more distant from the neighbors than males that produced type A notes with
more, short pulses within longer notes. For parameters related to B notes, only
Repetition Rate was incorporated into the model (R = 0.21; Adjusted R² = 0.18; F3,96 =
8.49; p < 0.0001; AICc: 92.50; AICc weight: 0.60), and it significantly predicted spacing
(Table 8). Perch height and number of males calling in the chorus were also important
predictors of intermale spacing: perch height was positively, while the number of males
was negatively, associated with the spacing (Tables 7 and 8).
Body size of males, measured as Snout Vent Length (SVL), was predicted by
three of the four retained PCs related to the parameters of A notes(R = 0.16; Adjusted
R²= 0.13; F3.96 = 6.11; p = 0.0007; AICc = -33.25; AICc weight = 0.451; Table 9), but just
the PC1 (Note Structure) and PC2 (Note Rate) were statistically significant predictors in
= 0.08; Adjusted R²= 0.06; F2.97 = 4.08; p = 0.02; AICc = -26.07; AICc weight = 0.304)
but only Note Structure predicted significantly males SVL (Table 10). In both cases, the
best fit models incorporated non-significant PCs.
The type of note broadcasted and the number of male calling each night had a
significant effect on the distance between the speaker and the nearest calling male,
while no effect of water body or the interaction between water body and the number of
males in the chorus was detect (Table 11). Calling males were closest to the speaker
broadcasting B notes than to the speaker broadcasting A notes (Figure 7): the mean
distance between speaker broadcasting A notes and nearest male was 2.49 ± 2.16 m,
while males around the speaker broadcasting B notes adopted calling sites at a
distance of 1.93 ± 0.95 m. An average of 11.03 ± 7.36 males was present in the chorus
each night. The total number of males in the chorus influenced the resulting intermale
spacing as short distances separated calling males when many were present, while
males were far apart when the chorus was formed by few males (Figure 8).
DISCUSSION
In this study, we investigated which advertisement call parameters and which
type of note convey information related to the maintenance of the intermale spacing
and to the competitive ability relative to male’s body size.
The characteristics of the studied water bodies influenced the number of calling
males, the intermale spacing and the perch heights adopted by D. nanus. In the lake, it
was registered an average number of calling males significantly greater than the
permanent and temporary ponds, but the highest number of males did not result in
shorter distances between calling males despite the expected decline of intermale
spacing with the increase of the number of calling males and density in the chorus
(Brenowitz et al. 1984, Gerhardt et al. 1989, Dyson & Passmore 1992, Shepard 2002).
Additionally, although the number of males in the permanent and temporary ponds did
smaller than at the permanent pond. These results may reflect (1) differences in the
size of water bodies: the shoreline area available for establishment of calling sites is
much larger at the lake than at the permanent pond, and the shoreline available for
occupancy at the permanent pond is larger than the area at the temporary pond or (2)
low availability of perches in the temporary pond for males establish calling sites. Since
males were found calling perched and also on the ground level, the availability of
perches did not seem to restrict the occurrence of calling males at each studied water
body, but where high-growing vegetation, such as shrubs and cattails, was available
(permanent water bodies), males were frequently found using these vegetation types
as calling sites and, therefore, calling from elevated positions. Calling from an elevated
position may enhance sounds propagation: when the signaler is elevated above the
ground, the attenuating effect of the ground surface is reduced, and the effective
distance that the sound may propagate is increased (e.g. Mitchell and Miller 1990,
Forrest 1994, Parris 2002). Thus, the area over which the call will effectively prevent a
closer approach by rivals is increased, and males will space themselves more widely.
Although the effects of the number of calling males and perch height on intermale
spacing varied with the water body characteristics, the results of model selection
indicate that these variables play an important role in determining spacing along with
acoustic parameters of both introductory (A) and secondary (B) notes.
Parameters of A notes that grouped in the first component (Note Structure) were
significantly related to intermale spacing, indicating their importance as predictors of
nearest-neighbor distance. Duration of notes, number and duration of pulses, and
pulses repetition rate are parameters related to the calling effort of signalers and may
indicate to competitors the male’s motivational state to fight, as emission of longer
notes by males is generally associated with aggressive interactions (e.g. Wells &
Schwartz 1984, Wells 1988, Lesbarrères and Lodé 2002). For example, Wells &
Schwartz (1984) verified that male of Hyla ebraccata present graded response to the
Besides, the production of notes of longer duration helps to increase the signal to noise
ratio of males vocalization, which make them more conspicuous to conspecifics (Wells
1988, Gerhardt 1992), indicating that the lengthening of the call in response to
competitors may function to balance the competition for mates and agonistic behaviors
(Wells 1988, Wagner 1989). However, though these researches suggest that
parameters related to the note structure mediate aggressive interactions and,
therefore, would be correlated to reduced territories, we found a negative relationship
between note structure and spacing, indicating that males who emit longer notes did
not hold larger territories and are closer to other males than its competitors who emit
shorter notes. Taking together with this finding the result of SVL modeling, which
indicates that A note structure is positively associated to body size, we verified that the
larger males were responsible for the production of the longer notes. As males with
larger body sizes may obtain more food than the smaller ones, which guarantee energy
enough to allow them to sustain longer duration of signaling (Gerhardt 1992). As high
calling effort involves the expenditure of great amount of energy (Ryan 1988), it is
expected that this calling behavior be associated with larger males. As males’ body size
can be assessed through the A note structure, other males in the chorus are allowed to
evaluate its ability to fight and win combats (Robertson 1986). If the larger male is a
superior competitor in comparison to the male doing the evaluation, then aggressive
attacks against it will be avoided. So, larger males may occupy suitable calling sites
independently of the closeness of other males, tolerating their presence while they
offered no threats to its position in the chorus. This may be a better strategy than
engage into a fight at the expense of energy which may be allocated to reproduction.
The second component of spacing being modeled by parameters of A note
(Note Rate) also was incorporated in the best fit model that explained intermale
spacing but it was not a statistically significant predictor. Note repetition rate is a
parameter related to the calling effort of males and may provide reliable information
indicate the physiological condition of the calling male (Gerhardt 1992, Schwartz 1994,
Schwartz 2001, Schwartz et al. 2002). Another acoustic parameter was grouped in this
component: the maximum frequency. Signals of low frequency propagate through
longer distances (Gerhardt 1994) and, for many species, are able to indicate callers
body size to coespecifics (Ramer et al. 1983, Robertson 1986). Therefore, it would be
expected that frequency should influence intermale spacing if not for its propagation
properties then for the information about the body size. However, the results of model
selection did not reveal any significant relationship among these acoustic parameters
and spacing. In relation to the body size, although call frequency is often negatively
associated with signaler size (Ramer et al. 1983, Robertson 1986), frequency
parameters were not consistently associated with body size in D. nanus. The only
frequency parameter with a significant association to SVL was the A note maximum
frequency in the PC2 (Note Rate). This parameter was negatively correlated with PC2,
while this PC was positively associated to SVL, indicating that a low maximum
frequency is related to a larger male. Other frequency parameters of A notes were
grouped in the third component of the SVL model which, although not significantly
associated to the SVL, presented a negative association with body size (i.e. high
values of minimum and fundamental frequencies indicate smaller male) and
contributed to the model increasing its predictive power. So, note repetition rate convey
information about the physiological condition, while maximum frequency informs about
body size of males. Probably these parameters are used by the females in the
assessment of the reproductive fitness of the male.
About the acoustic parameters of B notes, a positive association of parameters
grouped in the PC2 (Repetition Rate) with the intermale spacing indicate that the
distance between nearest calling males will be greater as the note and pulse repetition
rate increases. Note repetition rate is frequently adjusted as a result of male–male
competition in ways that may help to maintain or increase the male’s relative
repetition rate has the potential to transmit information about species and individual
identity (Gerhardt 1992) and also may mediate aggressive interactions (Wells 1988).
So, these parameters may be used by conspecific to assess which individual in the
chorus is calling and how high is its motivation to fight and breed, with males spacing
themselves as a way to increase their ability to attract females through the calling
activity and the properties of their calls. In relation to body size, only the PC1 (Note
Structure) was significantly and positively related to SVL, indicating that larger males
produced B notes with shorter and more numerous pulses than smaller males. In
accordance with what it was found to A notes acoustic parameters, also for B notes
frequency parameters did not encode information about body size. This result may
indicate that other acoustic parameters are more effective predictors of body size in D. nanus than is frequency.
Several researches have demonstrated that different advertisement call
parameters may convey different information about the signalers. Measures of
frequency may provide a reliable indicative of the body size, related to fight ability (e.g.
Ramer et al. 1983, Robertson 1986). Note repetition rate reflects, in aggressive
contexts, the motivation of the resident male to defend its territory or attack a
competitor, while in reproductive contexts, allows females to infer the reproductive
fitness of the signaling male since maintenance of high repetition rates may be costly to
the male (Gerhardt 1992, Welch et al. 1998, Schwartz 2001). SPL is a parameter that
may allow males to assess the distance of the signaler (e.g. Robertson 1984). The
pulse repetition rate is a static property of the call with the potential to transmit
information about specific and individual identity (Gerhardt 1992). Duration of notes
constitutes an honest indicator of male genetic quality potentially influencing patterns of
female mate choice (Welch et al. 1998). So, the verified association of parameters of
both note types A and B with intermale distance may reflect the different functions of
these parameters and notes in the establishment and maintenance of spacing. Also,
from a combination of parameters that reflect both notes structure. According to
Gerhardt (1992), if the values of different parameters are highly correlated, then such
redundancy may contribute to a more reliable extraction of the encoded information.
The relationship among acoustic parameters of A notes, spacing and body size
indicate that components related to the distance between calling males are also
important predictors of SVL of D. nanus. Both factor components included in the SVL
model were also included in the spacing model, but just one (Note Structure) was a
statistically significant predictor of SVL. Therefore, it seems that the same A note
parameters are responsible for conveying both kind of information in a way that
spacing may reflect not only the acoustic parameters and their propagation but also the
body size of calling males. It is possible to infer that the establishment of intermale
spacing at the beginning of the chorus, characterized by males emitting A notes, occurs
through the assessment of the competitive ability, relative to the body size, of rivals
encoded in the calls emitted. On the contrary, the parameters of B notes associated
with the spacing are not related to SVL, conveying just the information of location and
distance between competitors, while information about the body size is encoded in
other parameters. Therefore, in relation to spacing, parameters of A and B notes
convey different information: while the A note informs competitors about fight ability,
mainly related to males’ SVL, B note advertise the males reproductive fitness to
females.
The results of the playback experiment indicate that the type of note emitted
influences how males spaces throughout the reproductive habitat: males assumed
calling position farther from the speaker broadcasting A notes than the speaker
broadcasting B notes. So, although results of the advertisement call parameters
analysis indicate that both notes convey information that affects intermale spacing in D. nanus, the playback experiment reveals that A notes may be more effective in
determining spacing than B notes. This result confirm the hypothesis that D. nanus A
Considering a male-male interaction, it is possible that, at the beginning of chorus
activity, males emit A notes to inform their location and competitive ability to other male
who may use this information in the establishment of its calling site, ultimately resulting
in the establishment of intermale spacing, while emission of B notes during the chorus
activity may reinforce the message.
In summary, males of Dendropsophus nanus use acoustic signals to advertise
their body size and their location in the reproductive habitat to other conspecifics which
may assess the fighting ability and the distance of the calling male. This information is
transmitted through both notes that compose the advertisement call of this species.
Parameters of A notes convey both kinds of information, whereas parameters of B
notes affect male spacing but do not convey information about male body size.
Therefore, A notes appear to advertise mainly a male’s competitive ability in male-male
interactions, whereas B notes appear to advertise the males’ reproductive quality to
females and the different information conveyed by the different notes reflects in the
intermale distance. When the distance between calling males is mediated by A notes,
males occupy sites distant from each other, whereas they adopt close positioning in the
TABLES
Table 1. Mean ± Standard Deviation (SD) of type A and B notes parameters and results from paired t-test used to compare parameters between the notes. Parameters were considered significantly different between notes when p < 0.05.
Mean ± SD of notes Paired t-test Acoustic
Parameters A B t df p
Sound Pressure
Level (dB) 86.06 ± 5.20 87.46 ± 5.23 -13.93 99 < 0.0001 Fundamental
frequency (kHz) 4.24 ± 0.18 4.25 ± 0.17 0.82 99 0.412 Minimum
frequency (kHz) 2.10 ± 0.50 2.30 ± 0.50 -7.88 99 < 0.0001 Maximum
frequency (kHz) 21.89 ± 0.23 21.85 ± 0.35 1.50 99 0.138 Duration of note
(s)
0.038 ±
0.006 0.021 ± 0.004 33.17 99 < 0.0001
Note repetition
rate (notes/s) 0.81 ± 0.34 5.91 ± 0.59 -4.07 99 < 0.0001 Duration of pulses
(s) 0.003 ± 0.0006 0.004 ± 0.001 -11.59 99 < 0.0001
Number of pulses 13.57 ± 3.96 5.91 ± 1.60 24.04 99 < 0.0001
Pulses repetition
rate (pulses/s) 10.83 ± 5.59 5.91 ± 3.11 7.81 99 < 0.0001
Table 2. Correlations among nine acoustic parameters of type A notes. Pearson correlation coefficients are given below the diagonal and P values are given above the diagonal (two-tailed, n = 100). Statistically significant (p < 0.05) correlations are indicated in bold type.
Acoustic parameters SPL FF MinF MaxF DN NRR NPN DP PRR Sound pressure level (SPL) 0.95 0.03 0.05 0.65 0.98 0.50 0.40 0.60
Fundamental frequency (FF) -0.01 0.19 0.22 0.32 0.91 0.14 0.06 0.78
Minimum frequency (MinF) -0.22 0.06 0.78 0.10 0.86 0.01 0.02 0.19
Maximum frequency (MaxF) 0.20 0.03 -0.03 0.52 0.63 0.24 0.08 0.65
Table 3. Correlations among nine acoustic parameters of type B notes. Pearson correlation coefficients are given below the diagonal and P values are given above the diagonal (two-tailed, n = 100). Statistically significant (p < 0.05) correlations are indicated in bold type.
Acoustic parameters SPL FF MinF MaxF DN NRR NPN DP PRR Sound pressure level (SPL) 0.44 0.01 0.08 0.59 0.22 0.33 0.12 0.42
Fundamental frequency (FF) -0.08 0.12 0.25 0.76 0.09 0.14 0.02 0.31
Minimum frequency (MinF) -0.27 0.25 0.58 0.89 1.00 0.02 0.89 0.34
Maximum frequency (MaxF) 0.18 0.01 0.06 0.17 0.69 0.14 0.44 0.45
Duration of notes (DN) 0.05 -0.03 0.01 0.14 0.98 0.00 0.27 0.10
Notes repetition rate (NRR) 0.12 -0.17 0.00 0.04 0.00 0.04 0.05 0.00 Number of pulses/note (NPN) -0.10 0.15 -0.23 0.15 0.50 -0.21 0.00 0.13
Duration of pulses (DP) 0.16 -0.24 0.23 -0.08 0.11 0.20 -0.74 0.24
Pulses repetition rate (PRR) 0.08 -0.10 -0.10 0.08 0.17 0.85 0.15 -0.12
Table 4. Mean ± Standard deviation (SD) of 100 calling males’ perch height, intermale
spacing and total number per night.
Variables Permanent pond Lake Temporary pond
Number of recorded males 30 55 15
Perch height (m) 0.36 ± 0.17 0.39 ± 0.11 0.18 ± 0.10
Distance between calling males (m) 3.58 ± 3.92 2.74 ± 3.48 1.03 ± 0.56
Table 7. Multiple regression of the best fit model in which spacing is predicted by two of the retained PCs related to A notes emitted by males of D. nanus, perch height, and
number of males (N males). B: unstandardized coefficient; Beta: standardized coefficient; r: partial correlation coefficient.
B Std. Error Beta t(95) p r
Intercept 0.216 0.097 2.232 0.028
Note Structure (PC1) -0.066 0.023 -0.269 -2.944 0.004 -0.289
Note Rate (PC2) 0.050 0.030 0.151 1.670 0.098 0.168
Perch height 0.498 0.235 0.192 2.123 0.036 0.212
N males -0.009 0.003 -0.249 -2.725 0.008 -0.269
Table 8. Multiple regression of the best fit model in which spacing is predicted by one of the retained PCs related to B notes emitted by males of D. nanus, perch height, and
number of males (N males). B: unstandardized coefficient; Beta: standardized coefficient; r: partial correlation coefficient.
B Std. Error Beta t(96) p r
Intercept 0.169 0.099 1.707 0.091
Repetition Rate (PC2) 0.076 0.027 0.258 2.809 0.006 0.275
Perch height 0.702 0.238 0.271 2.948 0.004 0.288
N males -0.011 0.003 -0.296 -3.257 0.002 -0.315
Table 9. Best-fit model for the multiple regression of Snout Vent Length (SVL) on three of the retained PCs related to A notes emitted by males of D. nanus. B: unstandardized
coefficient; Beta: standardized coefficient; r: partial correlation coefficient.
B Std. Error Beta t(96) p r
Intercept 2.110 0.020 106.382 << 0.001
Note Structure (PC1) 0.038 0.012 0.301 3.222 0.002 0.312
Note Rate (PC2) 0.038 0.016 0.221 2.364 0.020 0.234
Frequency (PC3) -0.027 0.018 -0.144 -1.538 0.127 -0.155
Table 10. Best fit model for the multiple regression of Snout Vent Length (SVL) on two of the retained PCs related to B notes emitted by males of D. nanus. B: unstandardized
coefficient; Beta: standardized coefficient; r: partial correlation coefficient.
B Std. Error Beta t(97) p r
Intercept 2.110 0.021 102.03 << 0.001
Note structure (PC1) 0.034 0.014 0.232 2.380 0.019 0.234
Table 11. Effects of note type (A and B), number of males in the chorus, and water body where the experiment was conducted (fixed components) on the distance between speakers and the nearest calling male.
Value Std. Error df t p
Intercept 3.32 0.45 185 7.41 0.00
Note type -0.60 0.18 185 -3.30 0.001
Number of males -0.10 0.03 185 -3.01 0.003
Water Body -0.40 0.66 18 -0.61 0.551
FIGURES
Figure 1. (a) Oscilogram and (b) spectrogram of type A and B notes that compose the
Figure 2. Power spectrum of Dendropsophus nanus (a) type A and (b) B notes.
Figure 3. Two males Dendropsophus nanus showing the distinctive pattern of marks
Figure 4. Oscillograms of (a) a sequence of notes and (b) one Type A note emitted by
males of Dendropsophus nanus showing the parameters Notes Repetition Rate, Note
Figure 5. Spectrogram of a Type A note emitted by male Dendropsophus nanus,
showing the parameters measure Pulse Repetition Rate, Number of Pulses per Note
Figure 6. Power spectrum of a Type B note emitted by male of Dendropsophus nanus,
showing the parameters Fundamental Frequency (FF), minimum and maximum
frequency, and the bandwidth of frequencies.
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